Repair and/or reconstitution of invertebral discs

ABSTRACT

This invention relates to a method for repair and reconstitution of invertebral discs in a subject which involves administration of STRO-1 +  multipotent cells. The method of the invention is useful in the treatment of spinal conditions characterized by degeneration of the invertebral disc.

FIELD OF THE INVENTION

This invention relates to a method for repair and reconstitution ofinvertebral discs in a subject. The method of the invention is useful inthe treatment of spinal conditions characterized by degeneration of theinvertebral disc.

BACKGROUND OF THE INVENTION

The invertebral disc (IVD) is the largest predominantly avascular,aneural and alymphatic structure in the human body. The disc is criticalfor the normal function of the spinal column since it providesflexibility and mechanical stability during axial compression, flexionand extension. The IVD is composed of several specialised connectivetissues: (i) the hyaline cartilage of the cartilaginous end plates(CEPs) which cover the surface of the vertebral bones (bodies) which arepositioned above and below the disc; (ii) the fibrocartilagenous annulusfibrosus (AF) which encapsulates the nucleus pulposus (NP); and (iii)the central gelatinous nucleus pulposus (NP) which although it containscartilage like cells is not a hyaline cartilage. A transitional zone(TZ) has also been identified which, as its name implies, is locatedbetween the AF and NP. The fibrocartilagenous AF is composed ofconcentric collagenous layers (lamellae) that are connected to thebony-rim of the vertebral bodies.

Proteoglycans (PGs) and types I, II, III, V, VI, IX, X, XI collagens arethe major matrix components of all these disc tissues but their relativeabundance and distribution is dependent on their anatomical locations.PGs, which have a high affinity for water molecules, are most abundantin the NP of “healthy discs”. The water imbibed by the PGs generates ahydrostatic pressure within the NP that “inflates” the encapsulatingfibrocartilagenous AF. It is the combination of these specialisedconnective tissues with their individual physiochemical properties thatcontributes to the hydrodynamic and viscoelastic properties of the IVDthat are essential for the normal biomechanical function of the spinalcolumn.

The IVD undergoes profound matrix changes during ageing anddegeneration. Studies of human cadaveric and disc specimens obtained atthe time of spinal surgery have shown that discs from individuals in themiddle to older age groups generally have a wide range of lesions (1,2).

Three major types of disc lesions have been identified from thesespecimens: (i) the rim lesion, a transverse defect close to theattachment of the AF to the bone of the vertebral body rim; (ii) theconcentric (circumferential) tear, where the annular lamellae separatefrom each other; and (iii) the radiating tear which results from thepropagation of clefts initiating within the NP (1, 2, 3). Rim-lesionsare of particular interest since they appear more commonly inadolescence and early adult life within the anterior region of the AFclose to its insertion into the bone of the vertebral rim suggestingthat they may be mechanically mediated. Their presence suggests earlyfailure of the AF and is the primary cause of disc degeneration butstudies on cadaveric specimens also indicate that other pathologicalfeatures (concentric tears, cystic annular degeneration, dehydration ofthe NP, vertebral rim syndesmophytes and osteoarthritis of the posteriorintervertebral joints) are also invariably present to some degree (1,2). Although the temporal history of these respective disc lesions stillremains the subject of debate it is generally agreed that loss of PGsand its associated water from the NP is an early etiological determinantof disc degeneration (4).

As already discussed the disc functions as a flexible hydro elasticcushion, largely mediated by the imbibition of water molecules withinthe NP. A decline in water content and thus swelling pressure of the NPwould lead to the imposition of supraphysiological mechanical stresseson the AF resulting in localised failure.

Medical problems associated with back and neck-pain arising from discdegeneration are experienced by 90% of the population some time duringtheir lives (5, 6). In man, back or neck pain of sufficient severity towarrant medical intervention increases in incidence in the third andfourth decades of life, peaks in the fifties and declines thereafter(5).

In the USA, back pain is the second most common reason for visit to aphysician and medical conditions related to back and neck pain accountfor more hospitalisations than any other musculoskeletal disorder. Backpain is the primary cause of lost working hours. For example, in theUnited Kingdom it has been estimated that more than 11 million workingdays are lost annually from this complaint. Moreover, as the longevityof the population increases over the next few decades, back and neckpain problems are expected to increase accordingly.

Despite the high incidence and economic burden of neck and back pain inmodern societies, the causes are still poorly understood. There ishowever general agreement that degeneration and/or failure if the IVD isthe primary cause of pain, either directly from the nerves present inthe outer AF or from the adjacent spinal structures that becomemechanically compromised by the loss of disc hydroelastic function (7,8, 9, 10). Disc disease is responsible for 23-40% of all cases of lowback pain (11, 12). The outer AF is innervated and nerve fibres mayextend as deeply as its inner third, thus any pathological changes tothe outer AF may invoke pain (13, 14, 15).

The existing paradigm for treating back or neck pain of discal origin isempirical, directed either toward life-style changes or use ofanti-inflammatory/analgesic drugs to minimise the symptomatology or tosurgical intervention that may require resection of the degeneratetissues or spinal arthrodesis to restrict movement. Notwithstanding thewidespread use of spinal fusion for the relief of neck or low back painit is known that this is not a benign procedure since the mechanicalstress imposed on adjacent discs by the introduction of the rigidsegment across the disc space accelerates degenerate changes in adjacentdiscs which may become symptomatic at a later stage (16). Clearlyalternative methods of treatment are required.

Intra-discal administration of the protein, osteogenic protein-1 (OP-1)(Bone morphogenetic protein-7), has been reported to stimulate discmatrix repair following experimentally produced degeneration. Discdegeneration was produced in rabbits by prior injection of thedepolymerising enzyme chondroitinase ABC into the disc NP—the procedurebeing known as chemonucleolysis (17). Both NP and AF cells were found tobe far more efficient at re-establishing a functional matrix afterchemonucleolysis. Disc cells embedded in a normal dense extracellularmatrix were found to be largely un-responsive to the stimulatory effectsof OP-1 on PG synthesis (17).

Studies examining potential cellular therapies to achieve repair ofdegenerate canine and human IVDs using autologous chondrocytes have beenreported (18, 19). The cells used were the chondrocytes harvested fromhealthy NP of the same species and subsequently re-implanted into thedefect disc. The disadvantage of this approach is that the cells usedfor this purpose would need to be harvested from adjacent healthy discsor from other donors of the same species. Violation of the AF isrequired to obtain such cells and this process not only damages AFstructure but also the removal of viable cells from the NP wouldaccelerate degenerative changes in this tissue. Clearly this procedurewould have limited human application.

SUMMARY OF THE INVENTION

The present application describes for the first time the in vivo use ofSTRO-1⁺ multipotential cells to promote reconstitution of the nucleipulposi and annuli fibrosi of degenerate intervertebral discs. TheSTRO-1⁺ multipotential cells were derived form an allogeneic source andwere well tolerated in the animal models used in this study. Thissuggests that the STRO-1⁺ multipotential cells from donors can be grownin large numbers and developed as “off the shelf” products for thetreatment of degenerate intervertebral discs.

Accordingly, the present invention provides a method for reconstitutingand/or repairing an invertebral disc in a subject, the method comprisingadministering to the invertebral disc mesenchymal precursor cells(STRO-1⁺ multipotential cells) and/or progeny cells thereof.

In an embodiment of the present invention, the STRO-1⁺ multipotentialcells and/or progeny cells thereof are administered into the nucleuspulposus of the invertebral disc.

Preferably, the STRO-1⁺ multipotential cells are also TNAP⁺, VCAM-1⁺,THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ or any combination thereof.

The STRO-1⁺ multipotential cells and/or progeny cells thereof may bederived from an autogenic, allogeneic or xenogenic source. In oneembodiment, the cells are derived from an allogeneic source.

The method of the present invention may also comprise administering aglycosaminoglycan (GAG), such as, for example, hyaluronic acid(hyaluronan) (HA), chondroitin sulfate, dermatan sulfate, keratansulfate, heparin, heparin sulfate, to the invertebral disc. The GAG canbe administered in the same or different composition as the STRO-1⁺multipotential cells and/or progeny cells thereof.

It will be appreciated that the method of the invention may be performedon any vertebrate. For example, the subject may be a mammal such as ahuman, dog, cat, horse, cow, or sheep.

The method of the present invention may be used in the treatment orprevention of spinal conditions characterized by degeneration of theintervertebral disc such as low back pain, age-related changes of theintervertebral disc or spondylolysis.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the lumber spinal levels treatedwith STRO-1 cells in all sheep Groups.

FIG. 2. Radiologically determined Disc Height Index (DHI). X-Raydetermined mean±SEM Disc Height Index (DM) at baseline and 3 monthsafter Chondroitinase ABC induced degeneration (3 mth pre-STRO-1 cells)for the sheep group who received low dose (A) and high dose (B) STRO-1cells.

FIG. 3: MRI determined aggregate disc degeneration scores forchondroitinase ABC injected discs. Mean±SEM of MRI determined aggregatedisc degeneration scores after injection with chondroitinase ABC priorto treatment with low dose STRO-1 cells +HA or HA alone for 3 months (A)or 6 months (B).

FIG. 4: MRI determined aggregate disc degeneration scores forchondroitinase ABC injected discs. Mean±SEM of MRI determined aggregatedisc degeneration scores after injection with chondroitinase ABC priorto treatment with low dose STRO-1 cells +HA or HA alone for 3 months (A)or 6 months (B).

FIG. 5: Aggregate Histopathology Disc Degeneration Scores. Means ofaggregate histopathology disc degeneration scores at 3 months (A) and 6months (B) for low dose STRO-1 cells. #=significantly different fromcontrol p<0.001. Ω=from STRO-1⁺ cells p<0.01

FIG. 6: Aggregate Histopathology Disc Degeneration Scores. Means ofaggregate histopathology disc degeneration scores at 3 months (A) and 6months (B) for high dose STRO-1 cells. #=significantly different fromcontrol p<0.001. Ω=from STRO-1⁺ cells p<0.01

FIG. 7: Aggregate MRI Disc Degeneration Scores. Aggregate MRI discdegeneration scores for low dose STRO-1 cells at 3 months (A) and 6months (B). #=significantly different from control p<0.05. Ω=fromSTRO-1⁺ cells p<0.05

FIG. 8: Aggregate MRI Disc Degeneration Scores. Aggregate MRI discdegeneration scores for high dose STRO-1 cells at 3 months (A) and 6months (B). #=significantly different from control p<0.05. Ω=fromSTRO-1⁺ cells p<0.05

FIG. 9: Nucleus Pulposus (NP) Histopathology Degeneration Scores. Meansof NP histopathology degeneration scores for low dose STRO-1 cells at 3months (A) and 6 months (B).

FIG. 10: Nucleus Pulposus (NP) Histopathology Degeneration Scores. Meansof NP histopathology degeneration scores for high dose STRO-1 cells at 3months (A) and 6 months (B).

FIG. 11. Biochemically determined glycosaminoglycan (GAG) content forNucleus Pulposus. Mean±SD Biochemically determined glycosaminoglycan(GAG) content for the Nucleus Pulposus for Discs injected with Low doseor High dose STRO-1 cells for 3 months (A) or 6 months (B).#=significantly different from control (p<0.05).

FIG. 12. Radiologically determined Disc Height Index (DHI). A: X-raydetermined mean±SEM disc height index (DHI) for chondroitinase ABCinduced degenerate discs 3 and 6 months after injection with HA orHA+low dose STRO-1 cells. B: X-ray determined mean±SEM disc height index(DHI) for chondroitinase ABC induced degenerate discs 3 and 6 monthsafter injection with HA or HA+high dose STRO-1 cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION GeneralTechniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,stem cell biology, molecular genetics, immunology, immunohistochemistry,protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, CRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

As used herein, the terms “treating”, “treat” or “treatment” includeadministering a therapeutically effective amount of STRO-1⁺multipotential cells and/or progeny cells thereof sufficient to reduceor eliminate at least one symptom of the specified condition.

As used herein, the terms “preventing”, “prevent” or “prevention”include administering a therapeutically effective amount of STRO-1⁺multipotential cells and/or progeny cells thereof sufficient to stop orhinder the development of at least one symptom of the specifiedcondition.

STRO-1⁺ Multipotential Cells or Progeny Cells

As used herein, the phrase “STRO-1⁺ multipotential cells” shall be takento mean STRO-1⁺ and/or TNAP⁺ progenitor cells capable of formingmultipotential cell colonies.

STRO-1⁺ multipotential cells are cells found in bone marrow, blood,dental pulp cells, adipose tissue, skin, spleen, pancreas, brain,kidney, liver, heart, retina, brain, hair follicles, intestine, lung,lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, andperiosteum; and are capable of differentiating into germ lines such asmesoderm and/or endoderm and/or ectoderm. Thus, STRO-1⁺ multipotentialcells are capable of differentiating into a large number of cell typesincluding, but not limited to, adipose, osseous, cartilaginous, elastic,muscular, and fibrous connective tissues. The specificlineage-commitment and differentiation pathway which these cells enterdepends upon various influences from mechanical influences and/orendogenous bioactive factors, such as growth factors, cytokines, and/orlocal microenvironmental conditions established by host tissues. In oneembodiment STRO-1⁺ multipotential cells are non-hematopoietic progenitorcells which divide to yield daughter cells that are either stem cells orare precursor cells which in time will irreversibly differentiate toyield a phenotypic cell.

In another embodiment, the STRO-1⁺ multipotential cells are enrichedfrom a sample obtained from a subject, e.g., a subject to be treated ora related subject or an unrelated subject (whether of the same speciesor different). The terms ‘enriched’, ‘enrichment’ or variations thereofare used herein to describe a population of cells in which theproportion of one particular cell type or the proportion of a number ofparticular cell types is increased when compared with the untreatedpopulation.

In another embodiment, the cells used in the present invention expressone or more markers individually or collectively selected from the groupconsisting of TNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ orany combination thereof.

By “individually” is meant that the invention encompasses the recitedmarkers or groups of markers separately, and that, notwithstanding thatindividual markers or groups of markers may not be separately listedherein the accompanying claims may define such marker or groups ofmarkers separately and divisibly from each other.

By “collectively” is meant that the invention encompasses any number orcombination of the recited markers or groups of peptides, and that,notwithstanding that such numbers or combinations of markers or groupsof markers may not be specifically listed herein the accompanying claimsmay define such combinations or sub-combinations separately anddivisibly from any other combination of markers or groups of markers.

Preferably, the STRO-1⁺ cells are STRO-1^(bright) (syn. STRO-1^(bri)).Preferably, the STRO-1^(bright) cells are additionally one or more ofTNAP⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺ and/or CD146⁺.

In one embodiment, the STRO-1⁺ multipotential cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630.

A cell that is referred to as being “positive” for a given marker it mayexpress either a low (lo or dim) or a high (bright, bri) level of thatmarker depending on the degree to which the marker is present on thecell surface, where the terms relate to intensity of fluorescence orother marker used in the sorting process of the cells. The distinctionof lo (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being sorted. A cell that isreferred to as being “negative” for a given marker is not necessarilycompletely absent from that cell. This terms means that the marker isexpressed at a relatively very low level by that cell, and that itgenerates a very low signal when detectably labelled or is undetectableabove background levels.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker protein (forexample the antigen recognised by STRO-1) than other cells in thesample. For instance, STRO-1^(bri) cells produce a greater fluorescentsignal, when labelled with a FITC-conjugated STRO-1 antibody asdetermined by fluorescence activated cell sorting (FACS) analysis, thannon-bright cells (STRO-1^(dull/dim)). Preferably, “bright” cellsconstitute at least about 0.1% of the most brightly labelled bone marrowmononuclear cells contained in the starting sample. In otherembodiments, “bright” cells constitute at least about 0.1%, at leastabout 0.5%, at least about 1%, at least about 1.5%, or at least about2%, of the most brightly labelled bone marrow mononuclear cellscontained in the starting sample. In a preferred embodiment,STRO-1^(bright) cells have 2 log magnitude higher expression of STRO-1surface expression relative to “background”, namely cells that areSTRO-1⁻. By comparison, STRO-1^(dim) and/or STRO-1^(intermediate) cellshave less than 2 log magnitude higher expression of STRO-1 surfaceexpression, typically about 1 log or less than “background”.

As used herein the term “TNAP” is intended to encompass all isoforms oftissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In aparticularly preferred embodiment, TNAP as used herein refers to amolecule which can bind the STRO-3 antibody produced by the hybridomacell line deposited with ATCC on 19 Dec. 2005 under the provisions ofthe Budapest Treaty under deposit accession number PTA-7282.

In one embodiment the STRO-1⁺ multipotential cells are capable of givingrise to clonogenic CFU-F.

It is preferred that a significant proportion of the STRO-1⁺multipotential cells are capable of differentiation into at least twodifferent germ lines. Non-limiting examples of the lineages to which themultipotential cells may be committed include bone precursor cells;hepatocyte progenitors, which are multipotent for bile duct epithelialcells and hepatocytes; neural restricted cells, which can generate glialcell precursors that progress to oligodendrocytes and astrocytes;neuronal precursors that progress to neurons; precursors for cardiacmuscle and cardiomyocytes, glucose-responsive insulin secretingpancreatic beta cell lines. Other lineages include, but are not limitedto, odontoblasts, dentin-producing cells and chondrocytes, and precursorcells of the following: retinal pigment epithelial cells, fibroblasts,skin cells such as keratinocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal muscle, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells.

In another embodiment, the STRO-1⁺ multipotential cells are not capableof giving rise, upon culturing, to hematopoietic cells.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used. In another useful embodiment of the invention,cells of a non-human animal (or if the patient is not a human, fromanother species) are used.

The present invention also contemplates use of supernatant or solublefactors obtained or derived from STRO-1⁺ multipotential cells and/orprogeny cells thereof (the latter also being referred to as expandedcells) which are produced from in vitro culture. Expanded cells of theinvention may a have a wide variety of phenotypes depending on theculture conditions (including the number and/or type of stimulatoryfactors in the culture medium), the number of passages and the like. Incertain embodiments, the progeny cells are obtained after about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10passages from the parental population. However, the progeny cells may beobtained after any number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Media may be solid,liquid, gaseous or a mixture of phases and materials. Media includeliquid growth media as well as liquid media that do not sustain cellgrowth. Media also include gelatinous media such as agar, agarose,gelatin and collagen matrices. Exemplary gaseous media include thegaseous phase that cells growing on a petri dish or other solid orsemisolid support are exposed to. The term “medium” also refers tomaterial that is intended for use in a cell culture, even if it has notyet been contacted with cells. In other words, a nutrient rich liquidprepared for bacterial culture is a medium. A powder mixture that whenmixed with water or other liquid becomes suitable for cell culture maybe termed a “powdered medium”.

In an embodiment, progeny cells useful for the methods of the inventionare obtained by isolating TNAP⁺. STRO-1⁺ multipotential cells from bonemarrow using magnetic beads labelled with the STRO-3 antibody, and thenculture expanding the isolated cells (see Gronthos et al. Blood 85:929-940, 1995 for an example of suitable culturing conditions).

In one embodiment, such expanded cells (progeny) (preferably, at leastafter 5 passages) can be TNAP⁻, CC9⁺, HLA class I⁺, HLA class II⁻,CD14⁻, CD19⁻, CD3⁻, CDI1a⁻c⁻, CD31⁻, CD86⁻, CD34⁻ and/or CD80⁻. However,it is possible that under different culturing conditions to thosedescribed herein that the expression of different markers may vary.Also, whilst cells of these phenotypes may predominate in the expendedcell population it does not mean that there is a minor proportion of thecells do not have this phenotype(s) (for example, a small percentage ofthe expanded cells may be CC9⁻). In one preferred embodiment, expandedcells still have the capacity to differentiate into different celltypes.

In one embodiment, an expended cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 25%, more preferably at least 50%, of the cells are CC9+.

In another embodiment, an expanded cell population used to obtainsupernatant or soluble factors, or cells per se, comprises cells whereinat least 40%, more preferably at least 45%, of the cells are STRO-1⁺.

In a further embodiment, the expanded cells may express one or moremarkers collectively or individually selected from the group consistingof LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5,CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD 90, CD29, CD18, CD61,integrin beta 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R (STRO-2=Leptin-R), RANKL, STRO-1^(bright)and CD146 or any combination of these markers.

In one embodiment, the progeny cells are Multipotential Expanded STRO-1⁺Multipotential cells Progeny (MEMPs) as defined and/or described in WO2006/032092. Methods for preparing enriched populations of STRO-1⁺multipotential cells from which progeny may be derived are described inWO 01/04268 and WO 2004/085630. In an in vitro context STRO-1⁺multipotential cells will rarely be present as an absolutely purepreparation and will generally be present with other cells that aretissue specific committed cells (TSCCs). WO 01/04268 refers toharvesting such cells from bone marrow at purity levels of about 0.1% to90%. The population comprising STRO-1⁺ multipotential cells from whichprogeny are derived may be directly harvested from a tissue source, oralternatively it may be a population that has already been expanded exvivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified STRO-1⁺ multipotential cells,comprising at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or95% of total cells of the population in which they are present. Thislevel may be achieved, for example, by selecting for cells that arepositive for at least one marker individually or collectively selectedfrom the group consisting of TNAP. STRO-1^(bright), 3G5⁺, VCAM-1, THY-1,CD146 and STRO-2.

MEMPS can be distinguished from freshly harvested STRO-1⁺ multipotentialcells in that they are positive for the marker STRO-1^(bri) and negativefor the marker Alkaline phosphatase (ALP). In contrast, freshly isolatedSTRO-1⁺ multipotential cells are positive for both STRO-1^(bri) and ALP.In a preferred embodiment of the present invention, at least 15%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the administered cells havethe phenotype STRO-1^(bri), ALP⁻. In a further preferred embodiment theMEMPS are positive for one or more of the markers Ki67, CD44 and/orCD49c/CD29, VLA-3, α3β1. In yet a further preferred embodiment the MEMPsdo not exhibit TERT activity and/or are negative for the marker CD18.

The STRO-1⁺ multipotential cell starting population may be derived fromany one or more tissue types set out in WO 01/04268 or WO 2004/085630,namely bone marrow, dental pulp cells, adipose tissue and skin, orperhaps more broadly from adipose tissue, teeth, dental pulp, skin,liver, kidney, heart, retina, brain, hair follicles, intestine, lung,spleen, lymph node, thymus, pancreas, bone, ligament, bone marrow,tendon and skeletal muscle.

It will be understood that in performing the present invention,separation of cells carrying any given cell surface marker can beeffected by a number of different methods, however, preferred methodsrely upon binding a binding agent (e.g., an antibody or antigen bindingfragment thereof) to the marker concerned followed by a separation ofthose that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody-based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies or ligands may be attached to a solid support to allowfor a crude separation. The separation techniques preferably maximisethe retention of viability of the fraction to be collected, Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS. Methods for performing FACS will be apparent to the skilledartisan.

Antibodies against each of the markers described herein are commerciallyavailable (e.g., monoclonal antibodies against STRO-1 are commerciallyavailable from R&D Systems, USA), available from ATCC or otherdepositary organization and/or can be produced using art recognizedtechniques.

It is preferred that the method for isolating STRO-1⁺ multipotentialcells, for example, comprises a first step being a solid phase sortingstep utilising for example magnetic activated cell sorting (MACS)recognising high level expression of STRO-1. A second sorting step canthen follow, should that be desired, to result in a higher level ofprecursor cell expression as described in patent specification WO01/14268. This second sorting step might involve the use of two or moremarkers.

The method obtaining STRO-1⁺ multipotential cells might also include theharvesting of a source of the cells before the first enrichment stepusing known techniques. Thus the tissue will be surgically removed.Cells comprising the source tissue will then be separated into a socalled single cells suspension. This separation may be achieved byphysical and or enzymatic means.

Once a suitable STRO-1⁺ multipotential cell population has beenobtained, it may be cultured or expanded by any suitable means to obtainMEMPs.

In one embodiment, the cells are taken from the subject to be treated,cultured in vitro using standard techniques and used to obtainsupernatant or soluble factors or expanded cells for administration tothe subject as an autologous or allogeneic composition. In analternative embodiment, cells of one or more of the established humancell lines are used to obtain the supernatant or soluble factors. Inanother useful embodiment of the invention, cells of a non-human animal(or if the patient is not a human, from another species) are used toobtain supernatant or soluble factors.

The invention can be practised using cells from any non-human animalspecies, including but not limited to non-human primate cells, ungulate,canine, feline, lagomorph, rodent, avian, and fish cells. Primate cellswith which the invention may be performed include but are not limited tocells of chimpanzees, baboons, cynomolgus monkeys, and any other New orOld World monkeys. Ungulate cells with which the invention may beperformed include but are not limited to cells of bovines, porcines,ovines, caprines, equines, buffalo and bison. Rodent cells with whichthe invention may be performed include but are not limited to mouse,rat, guinea pig, hamster and gerbil cells. Examples of lagomorph specieswith which the invention may be performed include domesticated rabbits,jack rabbits, hares, cottontails, snowshoe rabbits, and pikas. Chickens(Gallus gallus) are an example of an avian species with which theinvention may be performed.

Cells useful for the methods of the invention may be stored before use,or before obtaining the supernatant or soluble factors. Methods andprotocols for preserving and storing of eukaryotic cells, and inparticular mammalian cells, are known in the art (cf., for example,Pollard, J. W. and Walker, J. M. (1997) Basic Cell Culture Protocols,Second Edition, Humana Press, Totowa, N.J.; Freshney, R. I. (2000)Culture of Animal Cells, Fourth Edition, Wiley-Liss, Hoboken, N.J.). Anymethod maintaining the biological activity of the isolated stem cellssuch as mesenchymal stem/progenitor cells, or progeny thereof, may beutilized in connection with the present invention. In one preferredembodiment, the cells are maintained and stored by usingcryo-preservation.

Administration and Compositions

The dosage of STRO-1⁺ multipotential cells or progeny thereof to beadministered may vary according to factors such as the disease state,age, sex, and weight of the individual. Dosage regimens may be adjustedto provide the optimum therapeutic response. For example, a single bolusmay be administered, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It may be advantageous toformulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to physically discrete units suited as unitary dosages forsubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

In one example, the dosage of STRO-1⁺ multipotential cells administeredis in the range of 0.1 to 4.0×10⁶ cells. The dosage may be, for example,0.5×10⁶ cells.

It is to be appreciated that the STRO-1⁺ multipotential cells and/orprogeny thereof may be processed into various forms (e.g. solutionsuspension, solid, porous, woven, non-woven, particulate, gel, paste,etc.) before being added to the disc space.

Numerous biologic and synthetic materials are contemplated forco-injection with the STRO-1⁺ multipotential cells and/or progenythereof into a nucleus pulposus to restore normal mechanical and orphysiological properties to a damaged intervertbral disc. For example,one or more natural or synthetic glycosaminoglycans (GAGS) ormucopolysaccharides, such as, for example, hyaluronic acid (HA),chondroitan sulfate, dermatan sulfate, keratin sulfate, heparin, heparinsulfate, galactosaminoglycuronglycan sulfate (GGGS), see previouschanges and others, including their physiological salts, may be injecteddirectly into the nucleus pulposus. It has been suggested that HA playsa role in the stimulation of endogenous HA synthesis by synovial cellsand proteoglycan synthesis by chondrocytes, inhibits the release ofchondrodegradative enzymes, and acts as a scavenger of oxygen freeradicals known to play part in cartilage deterioration. Chondroitinsulfate and glucosamine injectables have similarly been shown to blockthe progression of articular cartilage degeneration. Arguably, otherGAG's may provide similar protective or restorative properties havingtherapeutic value making them ideal candidates for injection into a discundergoing degenerative disc disease. Another valuable property of GAG'sis their strong ability to attract and retain water. Thus, it may beappropriate to mix GAG's with water or other aqueous materials to form aviscous gel that may then be injected into the space created fromaspiration of a nucleus pulposus, or alternatively, added to an existingnucleus pulposus as a supplement. Natural “hydrogels” can thereby beformed which are capable of filling space in three dimensions and actinglike packing materials that resist crushing and enable a disc toadequately absorb the shock associated with movement.

Synthetic hyaluronic gels such as, for example, Euflexxa®, (FerringPharmaceuticals) or Restylane®. (Q-Med Aktiebolag Co., Sweden) aresuitable for use in the present invention.

Examples of other injectable synthetic materials that may be used forco-administration include medical wade silicone, Bioplastique®. (solidsilicone particles suspended in polyvinylpyrrolidone carrier; UroplastyBV, Netherlands), Arteplast® (microspheres of polymethylmethacrylate(PMMA) suspended in gelatin carrier; Artcs Medical, USA), Artecoll®(smooth PMMA spheres suspended in bovine cartilage carrier; ArtepharmaPharmazeu Tische, GMBH Co., Germany). Further, synthetic hydrogelcompositions may be employed as a filler material to restore normalshape to a disc, thereby restoring normal bio-mechanical functions.

Antioxidants having known chondroprotective abilities are alsocandidates for injection into the nucleus pulposus. Examples of theseinclude tocophereol (vitamin E), superoxide dismutase (SOD), ascorbate(vitamin C), catalase and others. Further, amphiphilic derivatives ofsodium alginate and the like are also contemplated herein for injection.Additionally recombinant osteogenic protein-1 (OP-1) is a good candidatefor injection because of its ability to promote the formation of aproteoglycan rich matrix by nucleus pulposus and annulus fibrosus cells.

Use of synthetic injectables is also contemplated. These areparticularly applicable to situations where the primary goal is torestore bio-mechanical function to a disc. Hyaluronic acid alone or incombination with other glycosaminoglycans may be used as a carrier todeliver a biologically active material. In a preferred embodiment,Hyaluronic acid and or other GAGs is used as a carrier for STRO-1⁺multipotential cells or progeny cells thereof. The concentration andviscosity of the hyaluronic acid/GAG composition is routinely adjustedto suit a given purpose.

In another example, the STRO-1+ multipotential cells or progeny cellsthereof may be delivered in admixture with fibrin glue. When used hereinthe term “fibrin glue” refers to the insoluble matrix formed by thecross-linking of fibrin polymers in the presence of calcium ions. Thefibrin glue may be formed from fibrinogen, or a derivative or metabolitethereof, fibrin (soluble monomers or polymers) and/or complexes thereofderived from biological tissue or fluid which forms a fibrin matrix.Alternatively, the fibrin glue may be formed from fibrinogen, or aderivative or metabolite thereof, or fibrin, produced by recombinant DNAtechnology.

The fibrin glue may also be formed by the interaction of fibrinogen anda catalyst of fibrin glue formation (such as thrombin and/or FactorXIII). As will be appreciated by those skilled in the art, fibrinogen isproteolytically cleaved in the presence of a catalyst (such as thrombin)and converted to a fibrin monomer. The fibrin monomers may then formpolymers which may cross-link to form a fibrin glue matrix. Thecross-linking of fibrin polymers may be enhanced by the presence of acatalyst such as Factor XIII. The catalyst of fibrin glue formation maybe derived from blood plasma, cryoprecipitate or other plasma fractionscontaining fibrinogen or thrombin. Alternatively, the catalyst may beproduced by recombinant DNA technology.

The rate at which the clot forms is dependent upon the concentration ofthrombin mixed with fibrinogen. Being an enzyme dependent reaction, thehigher the temperature (up to 37° C.) the faster the clot formationrate. The tensile strength of the clot is dependent upon theconcentration of fibrinogen used.

When the fibrin clot is generated in the presence of hyaluronan itundergoes interactions and becomes interdigitated with the cross-linkedmatrix. This matrix is known to play a major role in tissue regenerationand performs cell regulatory functions in tissue repair [Weigel P H,Fuller G M, LeBoeuf R D. (1986) A model for the role of hyaluronic acidand fibrin in the early events during the inflammatory response andwound healing. J Theor Biol. 119: 219-34]. The dissolution rate ofhyaluronan is also prolonged in the HA-Fibrin matrix which could bebeneficial in prolonging the therapeutic effects of this GAG (Wadstrom Jand Tengblad A (1993) Fibrin glue reduces the dissolution rate of sodiumhyaluronate. Upsala J Med. Sci. 98: 159-167).

Several publications describe the use of fibrin glue for the delivery oftherapeutic agents. For example, U.S. Pat. No. 4,983,393 discloses acomposition for use as an intra-vaginal insert comprising agarose, agar,saline solution glycosaminoglycans, collagen, fibrin and an enzyme.Further, U.S. Pat. No. 3,089,815 discloses an injectable pharmaceuticalpreparation composed of fibrinogen and thrombin and U.S. Pat. No.6,468,527 discloses a fibrin glue which facilitates the delivery ofvarious biological and non-biological agents to specific sites withinthe body. However, the use of fibrin+hyaluronan+to promote chondrogenicdifferentiation of STRO-1+ allogeneic cells has not been describedpreviously.

The composition comprising STRO-1⁺ multipotential cells and/or progenycells thereof is “surgically added” to the disc space. That is, thematerial is added by the intervention of medical personnel, asdistinguished from being “added” by the body's natural growth orregeneration processes. The surgical procedure preferably includesinjection through a hypodermic needle, although other surgical methodsof introducing the collagen-based material into the disc may be used.For example, the material may be introduced into a disc by extrusionthrough a dilated annular opening, infusion through a catheter,insertion through an opening created by trauma or surgical incision, orby other means of invasive or minimally invasive deposition of thematerials into the disc space.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions. Indeed, the results presented herein showthat transplantation of allogeneic STRO-1⁺ multipotential cells in sheepwas well tolerated in the absence of immunosuppression.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. The cells may be encapsulated in acapsule that is permeable to nutrients and oxygen required by the celland therapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, the cells may be geneticallymodified to reduce their immunogenicity.

It will be appreciated that the STRO-1⁺ multipotential cells or progenythereof may be administered with other beneficial drugs or biologicalmolecules (growth factors, trophic factors). When administered withother agents, they may be administered together in a singlepharmaceutical composition, or in separate pharmaceutical compositions,simultaneously or sequentially with the other agents (either before orafter administration of the other agents). Bioactive factors which maybe co-administered include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPOXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine) as well aslocal anesthetics.

Genetically-Modified Cells

In one embodiment, the STRO-1⁺ multipotential cells and/or progeny cellsthereof are genetically modified, e.g., to express and/or secrete aprotein of interest, e.g., a protein providing a therapeutic and/orprophylactic benefit, e.g., insulin, glucagon, somatostatin,trypsinogen, chymotrypsinogen, elastase, carboxypeptidase, pancreaticlipase or amylase or a polypeptide associated with or causative ofenhanced angiogenesis or a polypeptide associated with differentiationof a cell into a pancreatic cell or a vascular cell.

Methods for genetically modifying a cell will be apparent to the skilledartisan. For example, a nucleic acid that is to be expressed in a cellis operably-linked to a promoter for inducing expression in the cell.For example, the nucleic acid is linked to a promoter operable in avariety of cells of a subject, such as, for example, a viral promoter,e.g., a CMV promoter (e.g., a CMV-IE promoter) or a SV-40 promoter.Additional suitable promoters are known in the art and shall be taken toapply mutatis mutandis to the present embodiment of the invention.

Preferably, the nucleic acid is provided in the form of an expressionconstruct. As used herein, the term “expression construct” refers to anucleic acid that has the ability to confer expression on a nucleic acid(e.g. a reporter gene and/or a counter-selectable reporter gene) towhich it is operably connected, in a cell. Within the context of thepresent invention, it is to be understood that an expression constructmay comprise or be a plasmid, bacteriophage, phagemid, cosmid, virussub-genomic or genomic fragment, or other nucleic acid capable ofmaintaining and/or replicating heterologous DNA in an expressibleformat.

Methods for the construction of a suitable expression construct forperformance of the invention will be apparent to the skilled artisan andare described, for example, in Ausubel et al (In: Current Protocols inMolecular Biology. Wiley Interscience, ISBN 047 150338, 1987) orSambrook et al (In: Molecular Cloning: Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).For example, each of the components of the expression construct isamplified from a suitable template nucleic acid using, for example, PCRand subsequently cloned into a suitable expression construct, such asfor example, a plasmid or a phagemid.

Vectors suitable for such an expression construct are known in the artand/or described herein. For example, an expression vector suitable forthe method of the present invention in a mammalian cell is, for example,a vector of the pcDNA vector suite supplied by Invitrogen, a vector ofthe pCI vector suite (Promega), a vector of the pCMV vector suite(Clontech), a pM vector (Clontech), a pSI vector (Promega), a VP 16vector (Clontech) or a vector of the pcDNA vector suite (Invitrogen).

The skilled artisan will be aware of additional vectors and sources ofsuch vectors, such as, for example, Invitrogen Corporation, Clontech orPromega.

Means for introducing the isolated nucleic acid molecule or a geneconstruct comprising same into a cell for expression are known to thoseskilled in the art. The technique used for a given organism depends onthe known successful techniques. Means for introducing recombinant DNAinto cells include microinjection, transfection mediated byDEAE-dextran, transfection mediated by liposomes such as by usinglipofectamine (Gibco, Md., USA) and/or cellfectin (Gibco, Md., USA),PEG-mediated DNA uptake, electroporation and microparticle bombardmentsuch as by using DNA-coated tungsten or gold particles (Agracetus Inc.,WI, USA) amongst others.

Alternatively, an expression construct of the invention is a viralvector. Suitable viral vectors are known in the art and commerciallyavailable. Conventional viral-based systems for the delivery of anucleic acid and integration of that nucleic acid into a host cellgenome include, for example, a retroviral vector, a lentiviral vector oran adeno-associated viral vector. Alternatively, an adenoviral vector isuseful for introducing a nucleic acid that remains episomal into a hostcell. Viral vectors are an efficient and versatile method of genetransfer in target cells and tissues. Additionally, high transductionefficiencies have been observed in many different cell types and targettissues.

For example, a retroviral vector generally comprises cis-acting longterminal repeats (LTRs) with packaging capacity for up to 6-10 kb offoreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of a vector, which is then used to integratethe expression construct into the target cell to provide long termexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simianimmunodeficiency virus (SrV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.56:2731-2739 (1992); Johann et al, J. Virol. 65:1635-1640 (1992);Sommerfelt et al, Virol. 76:58-59 (1990); Wilson et al, J. Virol.63:274-2318 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700; Miller and Rosman BioTechniques 7:980-990, 1989; Miller,A. D. Human Gene Therapy 7:5-14, 1990; Scarpa et al Virology 75:849-852,1991; Burns et al. Proc. Natl. Acad. Sci. USA 90:8033-8037, 1993).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for nucleic acid delivery. AAV vectors can be readilyconstructed using techniques known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos, WO 92/01070 andWO 93/03769; Lehkowski et al. Molec. Cell. Biol. 5:3988-3996, 1988;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter Current Opinion in Biotechnology 5:533-539, 1992; Muzyczka.Current Topics in Microbial, and Immunol. 158:97-129, 1992; Kotin, HumanGene Therapy 5:793-801, 1994; Shelling and Smith Gene Therapy 7:165-169,1994; and Thou et al, J Exp. Med. 179:1867-1875, 1994.

Additional viral vectors useful for delivering an expression constructof the invention include, for example, those derived from the pox familyof viruses, such as vaccinia virus and avian poxvirus or an alphavirusor a conjugate virus vector (e.g. that described in Fisher-Hoch et al.,Proc. Natl Acad. Sci. USA 56:317-321, 1989).

EXAMPLES Example 1 Experimental Design

Twenty four sheep received injections of 1.0 IU Chondroitinase ABC(cABC) (Seikagaku Corporation, Japan) into three adjacent lumbar discs(nominally L3-L4, L4-L5, and L5-L6) to initiate progressive discdegeneration. The remaining lumbar discs (nominally L1-L2 and L2-L3)were not injected with cABC and were considered normal controls. Fifteenweeks (±3 weeks) following administration of cABC, injections of STRO-1⁺multipotential cells at either a high or low dose (4×10⁶ or 0.5×10⁶cells, respectively) or ProFreeze™ NOA Freezing Medium (LonzaWalkersville Md.) mixed with an equal volume of Euflexxa® hyaluronicacid (Ferring Pharmaceuticals) were administered directly into thenucleus pulposus of the intervertebral discs (Table 1). Animals werenecropsied either 3 months or 6 months post-injection. FIG. 1 is aschematic representation of the spinal levels used for the study and theprotocol for the treatments.

TABLE 1 Study Design Summary Disc 15 ± 3 weeks Baseline Analysis atGroup N (nominal) before Baseline Day 0 Tx Sacrifice 1 n = 6 L1-L2 Noinjection No injection 3 months Histology/ biochemistry Low dose L2-L3No injection No injection 3 months Histology/ biochemistry 3 MonthsL3-L4 Chondroitinase STRO-1⁺ 3 months Histology/ cells 0.5 × 10⁶biochemistry L4-L5 Chondroitinase No injection 3 months Histology/biochemistry L5-L6 Chondroitinase HA and NAO 3 months Histology/biochemistry 2 n = 6 L1-L2 No injection No injection 3 months Histology/biochemistry High dose L2-L3 No injection No injection 3 monthsHistology/ biochemistry 3 months L3-L4 Chondroitinase STRO-1⁺ 3 monthsHistology/ cells 4 × 10⁶ biochemistry L4-L5 Chondroitinase No injection3 months Histology/ biochemistry L5-L6 Chondroitinase HA and NAO 3months Histology/ biochemistry 3 n = 6 L1-L2 No injection No injection 6months Histology/bio chemistry Low dose L2-L3 No injection No injection6 months Histology/ biochemistry 6 months L3-L4 Chondroitinase STRO-1⁺ 6months Histology/ cells 0.5 × 10⁶ biochemistry L4-L5 Chondroitinase Noinjection 6 months Histology/ biochemistry L5-L6 Chondroitinase HA andNAO 6 months Histology/ biochemistry 4 n = 6 L1-L2 No injection Noinjection 6 months Histology/ biochemistry High dose L2-L3 No injectionNo injection 6 months Histology/ biochemistry 6 months L3-L4Chondroitinase STRO-1⁺ 6 months Histology/ cells 4 × 10⁶ biochemistryL4-L5 Chondroitinase No injection 6 months Histology/ biochemistry L5-L6Chondroitinase HA and NAO 6 months Histology/ biochemistry

Example 2 Expansion of Immunoselected STRO-1⁺ Multipotential Cells

The STRO-1+ multipotential cells used for these experiments were derivedfrom French Sheep and prepared by Lonza (USA). Bone marrow (BM)aspirates were obtained from the sheep and BM mononucleiar cells (BMMNC)were prepared essentially as described previously (US 2005-0158289).

STRO-1+ multipotential cells were subsequently isolated using the STRO-3antibody by magnetic activated cell sorting as previously described(WO2006/108229).

Example 3 Radiology

Animals had lateral plain radiographs taken of the lumbar spine underinduction anaesthesia at the following time points: Day 0 (Injection ofcABC), Day of Test Article administration (15±3 weeks followinginduction of lumbar disc degeneration) and 3 months and 6 monthsfollowing implantation of the Test Article, Evaluation of theradiographs was undertaken by a blinded observer using an index ofintervertebral height (DHI) calculated by averaging the measurementsfrom the anterior, middle and posterior parts of the IVD and dividing itby the average of the adjacent intervertebral body heights as describedpreviously (24).

Example 4 Magnetic Resonance Imagine (MRI)

MRIs were undertaken of all sheep lumbar spines under inductionanaesthesia approximately 15 weeks following induction of discdegeneration by the injection of chondroitinase ABC and prior totreatment with the intradiscal injections of HA or high and low doseHA+STRO-1⁺ cells (Tx). Three and 6 months after treatments andimmediately prior to necropsy (Nx), spinal MRI was again undertaken onall sheep.

The imaging was carried out on either of two MRI scanners. MRIsperformed prior to administration of STRO-1⁺ cells used a 1.5 T SiemensVISION MRI scanner with Numaris 33G software. MRIs undertaken postadministration of STRO-1⁺ cells used a 1.5 Tesla Siemens AVANTRO MRIscanner with syngo B13 software. Localizing scans were followed bysagittal imaging of the lumbar and sacral spine in T1, T1 Gradient echo(T1_Flash), T2 and STIR weightings, followed by T2 weighted axialimaging of the 6 disc levels L6/S1 L5/L6, L4/L5, L3/L4, L2/L3, L1/L2.Image sequences were provided on CDs that displayed each scan as seriesof 12 sagittal images. The digitised images obtained for the 12 sagittalMRI acquired sections were review by two blinded qualified observerusing the Pferrmann et al classification criteria for disc degenerationscoring system (25) summarised in Table 2. The final scores correspondedto the average of the scores from the 2 blinded assessors.

TABLE 2 MRI Classification of Ovine Disc Degeneration Scoring Systemusing the Pferrmann grading system (25) Structure Inhomogenous Inhomo-Inhomo- Inhomo- Homogenous, with or without genous, genous, genous,bright white horizontal bands gray grey to black black 1 2 3 4 5Distinction of Nucleus & Annulus Clear Unclear Lost 1 2 3 SingleIntensity Hyper-intense, iso-intense to cerebrospinal fluid IntermediateHypointense 1 2 3 Height of Intervertebral Disc Normal to Normal toslightly moderately Collapsed Normal decreased decreased disc space 1 23 4

Example 5 Histopathological Analysis

The discal units to be processed for histological and biochemicalanalysis were each separated by cutting through the adjacent cranial andcaudal vertebral bodies close to the growth plates with a bone saw.These spinal segments were fixed en bloc in Histochoice® for 56 h anddecalcified in several changes of 10% formic acid in 5% Neutral BufferedFormalin for 2 weeks with constant agitation until completedecalcification was confirmed using a Faxitron HP43855A X-ray cabinet(Hewlett Packard, McMinnville, USA).

The decalcified specimens were processed by standard histologicalmethods for paraffin embedded and cutting. Paraffin sections 4 micronthick were mounted on Superfrost Plus glass microscope slides(Menzel-Glaser), dried at 85° C. for 30 min then at 55° C. overnight.The sections were deparaffinised in xylene (4 changes×2 min) andrehydrated through graded ethanol washes (100-70% v/v) to tap water. Onesection from all blocks prepared from the sagittal slices was stainedwith haematoxylin and eosin (H&E). The H&E stained histological sectionswere coded and reviewed to assess the extent of degeneration by anindependent blinded histopathologist using a published (26) four-pointsemi-quantitative grading system (see Table 3). Additional tinctorialstains including Alcian Blue counter-stained with Neutral Red were alsoused to identify the distribution and assembly of matrix components inthe disc sections.

TABLE 3 Grading system of histologic changes in lower lumbar discs (BEPbony end-plate, CEP cartilaginous end-plate) Grade Annulus fibrosisNucleus pulposus Cartilage end-plate Margins/subchondral bone 1 Intactlamellae Homogeneity Uniform thickness Even thickness of BEP Narrowinter-lamellar matrix Absence of clefting Intact attachment to boneLamellar bone only Intact annulus attachment Uniform calcification <⅕ ofDistinct junction with CEP Vessels only in outer ⅓ depth Few vascularintrusions into CEP Uniform cell distribution 2 Minor lamellar splittingand Minor clefting Minor cartilage thinning Slightly uneven BEPdisorganisation. Minor widening of Minor cell necrosis Small transversefissures Schmorl's nodes matrix Minor disorganisation of Minor posteriordisplacement Irregular thickening of Minimal remodelling of BEPattachment Rim lesion without of annulus calcified zone Small marginalosteophytes reparative reaction Minor chondrone formation Few invadingvascular channels Small chondrones 3 Moderate widening of matrixModerate clefting Marked cartilage thinning Moderately uneven BEPmoderate fissuring of attachment Moderate cell necrosis Markedthickening of calcified Vascularised Schmorl's nodes Radiating tears notinvolving outer Cystic degeneration zone Moderate trabecular thickening⅓ minimal chondroid metaplasia Posterior displacement within Manytransverse fissures Defect in bone lamellae Cystic degeneration Vesselsin annulus Many vascular channels Minimal fibrosis tissue in marrowoutewr and middle ⅓ rim lesion Centripetal extension of Many chondronesspaces with minor reparative reaction collagen Medium-size osteophytesModerate chondrone formation 4 Extensive lamellar disorganisationComplete loss of nucleus Total loss of cartilage Marked uneven BEPRadiating tears extending into outer Loose body formation Calcificationof residual Ossified Schmorl's nodes ⅓ Marked chondrone formationcartilage Large osteophytes Extensive chondroid metaplasia Widespreadfissuring Marked trabecular thickening Vessels in all zones Markedfibrosis of marrow spaces Rim lesion with marked reparative Cartilageformation reaction

Example 6 Biochemical Analysis of Fixed Disc Tissue

The annulus fibrosus (AF) and nucleus pulposus (NP) were carefullydissected from the processed decalcified disc tissues remaining afterthe central sagital slice was removed for the histological studies. Thisprocess was more difficult for grossly degenerate discs where thedemarcation boundary between the NP and AF was lost. Aliquots of thefinely diced NP and AF tissues were freeze dried to constant weight andtriplicate portions (1-2 mg) of the dried tissues were hydrolysed in 6MHCl at 110° C. for 16 h. Aliquots of the neutralised digests assayed forhydroxyproline as a measure of the tissue collagen content as describedpreviously (4). Triplicate portions of freeze dried tissues (˜2 mg) werealso digested with papain and aliquots assayed for sulphatedglycosaminoglycan (GAGs) using the metachromatic dye1,9-dimethylmethylene blue as described previously (27).

Example 7 Statistical Analysis of Data

The statistical comparison of the DHIs and the biochemical data for allthe 3 and 6 month treatment groups was undertaken using the Student'sunpaired t-test where p<0.05 was considered significant. For thehistological and MRI aggregate degeneration and NP scores, comparisonbetween the various disc treatments was undertaken using theKruskal-Wallis or Friedman Tests (nonparametric repeated measures ANOVA)with Dunn's multiple comparison post hoc test. Statistical significancebetween groups was taken as p<0.05.

Example 8 Results of the Ovine Disc Re-Generation Studies UsingImmunoselected STRO-1⁺ Multipotential Cells

All animals in the high dose (4.0×10⁶ STRO-1⁺ cells) (Groups 2 and 4)and low dose (0.5×10⁶ STRO-1⁺ cells) (Groups 1 and 3) injected groupsmaintained normal body weights and showed no evidence of adverse sideeffects over the duration of the experiment.

The injection of the chemonucleolytic agent, chondroitinase ABC, intothe NP of target discs resulted in an approximate 50% decrease in discheight index (DHI) over 3 months as shown in FIG. 2; confirming asignificant loss of PGs and water from the disc extra-cellular matrix.The DHI pre-treatment data was supported by the MRI aggregate discdegeneration scores. As shown in FIGS. 3 and 4, for all groups, thenon-chondroitinase ABC injected control discs afforded MRI degenerationscores that were approximately 50% of the chondroitinase ABC injecteddisc scores confirming the validity of the model.

The aggregate of the mean histopathological grading scores determinedfor the discs of animals sacrificed 3 months after injection with lowdose STRO-1⁺ cells showed a decline in scores which were notsignificantly different to the non-injected control disc nor the cABC orcABC+HA injected discs. However, the latter 2 scores were significantlyhigher (p<0.001) than control disc scores (FIG. 5A). For the low doseSTRO-1⁺ cells group that was sacrificed 6 months post treatments thedisc scores were significantly lower (p<0.01) than the cABC and cABC+HAinjected discs but were equivalent to the non-injected control discscores (FIG. 5B). This pattern was maintained for the high dose STRO-1⁺cells discs at 3 months (p<0.05) (FIG. 6A) but not 6 months postinjection (FIG. 6B).

Photomicrographs of histological sections of non-injected control, cABC,cABC+HA and cABC+STRO-1⁺ cells injected discs of a 3 months low dosesheep and 6 months low dose sheep together with their respectivehistopathological scores were analysed. This analysis highlighted themarked structural and cellular variations resulting from the variousintra-discal treatments post cABC administration. It also illustratedthe beneficial effects mediated by the STRO-1⁺ cells as demonstrated bythe normalization of disc structural integrity and deposition of a newextracellular matrix 6 months following administration of the low doseof cells. Although these histological sections were selected on thebasis of their diverse histopathological scores, they were consistentwith the overall mean aggregate scores obtained for all the 3 and 6months low dose groups summarized in FIG. 5.

The disc histopathology scores were complimentary with the MRI assesseddisc degeneration scores. Moreover, in all groups the MRI scoresfollowed the same pattern of higher scores for the cABC alone andcABC+HA injected discs than for the cABC+STRO-1⁺ cells and control discsscores irrespective of the dose used. However, for the MRI degenerationscores, significant differences were observed for both the 3 and 6months low dose STRO-1⁺ cells injected disc scores relative to thecABC+HA scores (p<0.05) (FIGS. 7A &7B). MRI scores for high doseinjected STRO-1⁺ cells were not significantly different from control,cABC alone or cABC+HA values at 3 months but were less than cABC aloneat 6 months (FIGS. 8A and 8B).

Since the experimental model of disc degeneration used in theseexperiments was induced by the injection of the PG depolymerisingenzyme, chondroitinase-ABC, directly into the NP, the histolopathologyscores for this region are shown separately. In addition the biochemicalchanges that occurred in response to the various treatments for the NPtissues are also presented. As is evident from FIGS. 9 and 10 the meanNP histopathology scores for the cABC+STRO-1⁺ cells injected discsfollowed the same pattern as the overall disc histopathology scores withthe cABC and cABC+HA injected discs exhibiting higher degenerationscores. However, differences were not statistically significant due tointer-animal sample variation within the small group sizes used (N=6).

The radiologically determined disc height index (DHI) is a validatedindex of disc degeneration (24). As already discussed DHI were reducedby about 50% three months following intra-discal injection of the PGdepolymerising enzyme, cABC.

Biochemical determination of the glycosaminoglycan (GAG) content (as amarker of PGs) of the disc NP tissues showed a substantial loss in thiscomponent for all treated discs relative to the non-injected controldiscs (FIG. 11). While significant differences (p<0.05) in GAG levelswere observed for the cABC and cABC+HA tissues relative to control NPtissues for all groups, high dose STRO-1⁺ cells injected discs at 3months and low dose STRO-1⁺ cells at 6 months were not statisticallydifferent to controls (FIG. 11).

As can be seen from FIG. 12 all injected discs, irrespective of theirtreatments showed some recovery in their DHI over the 3 months of thepost treatment period. However, the largest improvement in DHI was notedfor discs injected with low dose STRO-1⁺ cells where the improvement wasshown to be statistically significant (p<0.02). Moreover, the DHI of thelow dose STRO-1⁺ cells injected discs at 6 months were not significantlydifferent from the non-injected control disc DHI mean values (FIG. 12A).

DISCUSSION

The results of the present experiments have shown that intra-discaladministration of low dose STRO-1⁺ cells +HA into degenerate discsimproved structural restoration to a greater extent than discs injectedwith HA. This conclusion was supported by the experimental datagenerated by three independent assessments of disc integrity and matrixrecovery from a baseline degenerative status corresponding to 50% ofnormal disc height index.

The increase in DHI observed for the low dose STRO-1⁺ cells injecteddiscs over 6 months may be explained by the deposition within thedegenerate discs of a reconstituted extra-cellular matrix. In thehealthy spinal column the disc height (DHI) is maintained by thepresence within the nucleus pulposus and inner-annulus of highconcentrations of PGs and their bound water molecules (2,4). Theseentities confer a high swelling pressure to the disc that maintains discheight but also allows the disc to recover from deformation after axialcompression (2,4). Indeed, the use of chondroitinase-ABC to induce discdegeneration in this animal model relied on the ability of this enzymeto selectively degrade and remove the majority of the PGs from theextra-cellular matrix of the NP and inner AF (28). The histochemicalstudies using the Alcian Blue dye, which binds to the negatively chargedPGs, showed for sections obtained from disc injected with low doseSTRO-1⁺ cells, more intense staining than the cABC alone of cABC+HAinjected disc sections confirming the presence of higher concentrationsof PGs in these tissues. Examination of these stained sections by bothwhite and fluorescent light microscopy also confirmed the lamellastructure of the AF collagen fibril assembly as well as thenormalization of hyaline cartilaginous end plate (CEP) morphology. TheCEP is a major route of nutrient diffusion into the avascular NP andphysical disruption of its structure diminishes the survival of theresident cell.

The concentrations of PGs in the NP, as determined biochemically fromthe GAG content, were only partly supportive of the histopathologyfindings. The GAG content of the high and low dose STRO-1⁺ cellsinjected NPs at 3 and 6 months were found not to be statisticallydifferent from control NP, while the other treated discs were. However,statistical differences between the GAG levels in the cABC+STRO-1⁺ cellsinjected discs and the cABC or cABC+HA disc NPs could not bedemonstrated suggesting comparable levels of GAGs within the tissues ofthese groups. As the disc tissues used for the biochemical analysis hadbeen previously fixed in buffered formalin for some months prior tobiochemical analysis it is possible that variable amounts of PGs leachedout of the matrices over this time. In addition, the collagen andprotein covalent crosslinks formed during the formalin fixation processmay have impaired adequate macroscopic distinction and dissection of theNP and AF regions of the disc from each other. The dissection wasparticularly difficult for degenerate discs where the boundary betweenNP and AF had been lost. Disc AF has a much lower GAG content than NPand therefore sampling errors could have a marked effect on theanalytical data (1-4).

Administration of ovine STRO-1⁺ multipotential cells together with asuitable carrier, such as high molecular weight hyaluronic acid (HA),into the nucleus pulposus of experimentally created degenerate IVDs hasbeen shown in the present experiments to accelerate the regeneration ofthe disc extracellular matrix as assessed radiographically by therecovery of disc height. This interpretation is based on the assumptionthat in the loaded spinal column the disc height is maintained by thepresence within the NP and inner-annulus of high concentrations ofmatrix proteoglycans that together with their bound water moleculesconfer a high swelling pressure to this structure. Indeed, the use ofchondroitinase-ABC to induce disc degeneration at the commencement ofthese experiments relied on the ability of this enzyme to degrade andremove the majority of the proteoglycans from the NP extracellularmatrix.

The present results indicated that the recovery of discs from thedegenerative state induced by earlier injection of cABC was moresustained with the lower dose of (0.5×10⁶) than with the higher dose(4.0×10⁶) of STRO-1⁺ cells. A possible explanation for this observationcould be related to the poor nutritional supply to the NP of the discthat is dependent on the exchange of O₂/CO₂ and metabolites between theblood vessels beneath to the CEP (1, 2). As already mentioned even minordisruption of the interface between the NP the CEP and the subchondralblood supply of the vertebrae, as was seen to occur in the degeneratediscs would be expected to impair nutrition to the NP. This nutritionaldeficiency could present an upper limit to the number of injectedSTRO-1⁺ cells that could survive in this oxygen-deprived environmentthereby resulting in loss of their viability and thus therapeuticbenefits.

The therapeutic mechanisms responsible for the recovery of discintegrity in this animal model following administration of low doseSTRO-1⁺ multipotential cells have yet to be resolved. However, it ispossible that anti-inflammatory cytokines and growth factors, releasedby the STRO-1⁺ multipotential cells within the degenerate disc spacewould modulate the pro-catabolic and anabolic suppressive effectsmediated by cytokines and other noxious factors released by the residentdisc cells in response to biochemical and biomechanical injury. TheseSTRO-1⁺ multipotential cells derived paracrine factors could alsosupport the normal physiological anabolic response of resident disccells to the depletion of PGs from their extra-cellular environment aswas occasionally seen in some of the non-STRO-1⁺ cells injected discs.On the other hand it is possible that some STRO-1⁺ multipotential cellsmay engraft within the degenerate matrix and undergo differentiationinto NP chondrocytes or other disc cells.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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1. A method for reconstituting and/or repairing an invertebral disc in asubject, the method comprising administering to the invertebral discSTRO-1⁺ multipotential cells and/or progeny cells thereof.
 2. The methodaccording to claim 1 wherein the STRO-1⁺ multipotential cells and/orprogeny cells thereof are administered into the nucleus pulposus of theinvertebral disc.
 3. The method according to claim 1 wherein the STRO-1⁺multipotential cells are also positive for TNAP+ (STRO-3), VCAM-1+,THY-1+, STRO-2+, CD45+, CD146+, 3G5+ or any combination thereof.
 4. Themethod according to claim 1 wherein the STRO-1⁺ multipotential cells arederived from an allogeneic source.
 5. The method according to claim 1which further comprises administering to the invertebral disc aglycosaminoglycan (GAG).
 6. The method according to claim 5 wherein theGAG is selected from the group consisting of hyaluronic acid (HA),chondroitan sulfate, dermatan sulfate, keratan sulfate, heparin, heparinsulfate, and galactosaminoglycuronglycan sulfate (GGGS).
 7. The methodaccording to claim 1 wherein the subject has a spinal conditioncharacterized by degeneration of the intervertebral disc.
 8. The methodaccording to claim 7 wherein the spinal condition is low back pain,age-related changes of the intervertebral disc or spondylolysis.